Figure 6. TTP inhibits NF-κB nuclear translocation.

5 × 10⁶ MDA-MB-231cells were infected with TTP/Ad for 24 hours and then stimulated with or without 5 ng/ml of TNF-α for 30 min, followed by nuclear and cytoplasmic protein extraction to measure TTP, p65, p50 and c-Rel by Western Blot with their respective antibodies A. Image represents one of three experiments with similar results. 5 × 10⁶ TTP^−/− and WT MEF cells were treated with or without 30 ng/ml TNF-α for 1 h and 3 h, followed by nuclear and cytoplasmic protein extraction to measure TTP, p65 and c-Rel by Western Blot. β-actin and PU.1 were used as control for cytoplasmic and nuclear protein B. 5 × 10⁶ HEK293 cells were transiently transfected with c-Jun promoter luciferase construct along with TTP and NF-κB expression vectors, followed by measurement of luciferase activity by luminometer C. The data shown were normalized to the results obtained from CMV empty co-transfection group. Results shown are mean plus SD of four independent experiments. The transfected cells of each condition were lysed for Western blot to determine the levels of TTP, NF-κB p65, c-Rel and p50 protein (lower panel in C). 5 × 10⁶ HEK293 cells were transiently transfected with c-Jun promoter construct along with IκB-α mutant and its empty vector PCR3.1, followed by measurement of luciferase activity by luminometer D. Results shown are mean plus SD of three independent experiments.